Pharmacovirological impact of an integrase inhibitor on human immunodeficiency virus type 1 cDNA species in vivo

Abstract

Clinical trials of the first approved integrase inhibitor (INI), raltegravir, have demonstrated a drop in the human immunodeficiency virus type 1 (HIV-1) RNA loads of infected patients that was unexpectedly more rapid than that with a potent reverse transcriptase inhibitor, and apparently dose independent. These clinical outcomes are not understood. In tissue culture, although their inhibition of integration is well documented, the effects of INIs on levels of unintegrated HIV-1 cDNAs have been variable. Furthermore, there has been no report to date on an INI's effect on these episomal species in vivo. Here, we show that prophylactic treatment of transgenic rats with the strand transfer INI GSK501015 reduced levels of viral integrants in the spleen by up to 99.7%. Episomal two-long-terminal-repeat (LTR) circles accumulated up to sevenfold in this secondary lymphoid organ, and this inversely correlated with the impact on the proviral burden. Contrasting raltegravir's dose-ranging study with HIV patients, titration of GSK501015 in HIV-infected animals demonstrated dependence of the INI's antiviral effect on its serum concentration. Furthermore, the in vivo 50% effective concentration calculated from these data best matched GSK501015's in vitro potency when serum protein binding was accounted for. Collectively, this study demonstrates a titratable, antipodal impact of an INI on integrated and episomal HIV-1 cDNAs in vivo. Based on these findings and known biological characteristics of viral episomes, we discuss how integrase inhibition may result in additional indirect antiviral effects that contribute to more rapid HIV-1 decay in HIV/AIDS patients.

Figures

FIG. 1.

Chemical structure and antiviral activity of the INI GSK501015. (A) Structure of the two-metal-binding naphthyridinone derivative GSK501015. (B to E) Dose-response curves for the antiviral potency of the INI GSK501015 in ex vivo cultures of primary T cells and macrophages from hCD4/hCCR5 transgenic rats and humans. (B and C) Activated T cells from both species. (D) Spleen-derived macrophages from double-transgenic rats. (E) Monocyte-derived macrophages from human donors were pretreated with the INI and subsequently challenged with HIV-1R7/3 YU-2 Env GFP (T cells) or VSV-G HIV-1NL4-3 GFP (macrophages). On day 3 postinfection, the percentage of GFP-positive cells was scored by flow cytometry, and the values obtained for untreated controls were set to 100%. Given are arithmetic means ± standard deviations (n = 3) of one experiment. EC50s were determined by using Prism software (GraphPad, San Diego, CA) and are shown in panels B to E and in Table 3.

FIG. 2.

Establishment of a highly sensitive and specific rat integration PCR and of basic pharmacovirological parameters of GSK501015 in rats. (A) Strategy of the “optimized protocol” for the nested rat integration PCR (see Materials and Methods for details). ID, rodent identifier consensus sequence. (B) The sensitivities of detection of HIV-1 integrations in the rat genome in a recently reported nested PCR protocol (standard protocol [19]) and the optimized protocol developed here were compared. Shown are the relative intensities of the specific signals (left), the percentage of unspecific signal (middle), and the lower limit of the standard (right), applying the two PCR protocols to the Rat2int genomic standard. (C) GSK501015 potently suppresses HIV-1 integrant formation in a rat cell line. Rat2 cells were pretreated with different concentrations of GSK501015 in the presence (1 μM) (negative control) or absence of the RTI efavirenz and challenged with a VSV-G-pseudotyped HIV-1 GFP vector. On day 7 postinfection, DNA was extracted from passaged cells, and the relative levels of HIV-1 integrants were determined as described in Materials and Methods. The number of integrants in the absence of treatment was set to 100%, and the relative percentages in the presence of decreasing concentrations of GSK501015 are depicted. (D) Relationship of oral dose and serum concentration of suspension-formulated GSK501015 in outbred, nontransgenic Sprague-Dawley rats. Data points indicate arithmetic means ± standard deviations (n = 3 animals).

FIG. 3.

Experimental design of the two in vivo studies with GSK501015 in hCD4/hCCR5-transgenic rats. (A) High-dose study. Transgenic rats were treated with GSK501015 at 10 mg/kg by twice-daily oral gavage for 5 days (GSK501015 group; n = 6) or left untreated (control group; n = 6). On day 1, the animals, including one hCD4 single-transgenic rat (ID 940), were challenged with HIV-1YU-2 by tail vein injection. On day 4 postinfection, the animals were sacrificed and their spleens were removed for the quantitative analysis of HIV-1 cDNA species. (B) Dose titration study. hCD4/hCCR5 transgenic rats were treated with GSK501015 at the indicated doses for 5 days or left untreated. On day 1, the animals, including one nontransgenic rat and one hCCR5 transgenic rat, were challenged with HIV-1YU-2 by tail vein injection. On day 4 postinfection, the animals were sacrificed and their spleens were removed for the quantitative analysis of HIV-1 cDNA species. Blood samples for concurrent pharmacokinetic analysis were taken from all INI-treated animals on the indicated days at −2 h, +2 h, and +6 h relative to the morning dosing.

FIG. 4.

High-dose treatment with GSK501015 inhibits HIV-1 integrant formation and elevates levels of episomal HIV-1 cDNA in vivo. The impact of INI treatment (10 mg/kg per day; the experimental design is shown in Fig. 3A) on HIV-1 DNA species abundance was assessed by determining, by real-time PCR, the load of HIV-1 integrants (A) or episomal two-LTR circles (B) relative to a rat GAPDH standard in DNA extracts from splenocytes. The results given for individual animals (left) are arithmetic means plus the standard error of the mean (SEM). The results given for animal groups (right) are geometric means + SEM. Nonparametric statistical analyses were performed by using the Mann-Whitney U test: integrants, 66-fold reduction (i.e., 98.5% reduction; P = 0.026); two-LTR circles, 7-fold increase (P = 0.03), n.a., not analyzed; ↓, no signal detected. *, statistically significant.

FIG. 5.

Concentrations of GSK501015 in sera from HIV-infected hCD4/hCCR5 transgenic rats. Blood samples were taken at the indicated time points relative to the morning dose administration, and GSK501015 concentrations were determined by liquid chromatography-tandem mass spectrometry. The dotted line indicates the in vitro PA-EC50 as an informative reference. p.i., postinfection.

FIG. 6.

Concentration-dependent efficacy of GSK501015 in HIV-infected hCD4/hCCR5 transgenic rats. These analyses contain results obtained from both the high-dose and the dose titration studies. (A) Number of HIV-1 integrants per ng DNA determined in the spleen versus the day 1 maximum concentration of GSK501015 (Cmax) in serum. The dotted curve indicates the fit of the Hill curve. (B) Levels of episomal two-LTR circles relative to the administered GSK501015 dose. The gray boxes indicate the geometric means of two-LTR circles in samples from the untreated control groups and the group treated at 10 mg/kg (P = 0.0172). The dotted line indicates the fitted linear relationship between log(two-LTR circles) and log dose.

FIG. 7.

In vivo relationship of HIV-1 integrants and two-LTR circles in a secondary lymphoid organ of GSK501015-treated rats. Data obtained from the two in vivo studies for levels of integrants and two-LTR circles in untreated or GSK501015-treated hCD4/hCCR5 transgenic rats are given (and also presented in part in Fig. 4 and 6). Each circle depicts the result for these two virological parameters for one HIV-infected rat (open circles, high-dose study; filled circles, dose titration study). The fitted line illustrates the inverse correlation determined by Pearson's correlation coefficient between log(integrants) and log(two-LTR circles).